Structural and sequence analysis of FAM155/Mid1 homologues. (A) Structural superimposition of the solved human NALCN–FAM155A cryo-EM structure (from PDB accession no. 7SX4) and predicted NALCN–FAM155 or Cch1-Mid1 dimer structures from the early-diverging animal T. adhaerens, the pathogenic fungus C. neoformans, and the Cryptista (algal) species C. curvata and G. cryophila. (B) Close-up view of the FAM155/Mid1 NALCN/Cch1 subunit interfaces (corresponding to the dashed black boxes in panel A, revealing similar positioning of predicted α1 to α3 helices, that in the human complex, form critical contacts with the NALCN subunit. Versions of these predicted structures, colored according to pLDDT confidence scores, are provided in Fig. S6 A. (C) Partial protein alignment of selected FAM155/Mid1 homologues from animals, fungi, apusomonads, and cryptists, revealing strong sequence conservation within the α1 to α3 helices. The chevrons above the alignment denote amino acids in the human FAM155A shown to make important contacts with NALCN in cryo-EM structures. Numbers to the right of the alignment denote amino acid positions. The boxes are used to label amino acids predicted to form helical structures by AlphaFold.